Photodynamic therapy (PDT), now a well recognized treatment for the destruction of tumors, utilizes the ability of a selectively retained photosensitizer to elicit an efficient photodynamic reaction upon activation with tissue penetrating light Pandey, R., Zheng, G. Porphyrins as Photosensitizers in Photodynamic Therapy, in The Prophyrin Handbook (Eds: Smith, K. M., Kadish, K., Guilard, R.) Academic Press, San Diego, 2000 Vol. 6; Sherman, W. M., Allen, C. M., van Lier, J. E., Role of Activated Oxygen Species in Photodynamic Therapy, Methods in Enzymology, 319, 376-400, 2000). Though a large number of porphyrin based photosensitizers have been reported since the introduction of the first PDT drug Photofrin®, there has not been much success on improving the photosensitizer's tumor selectivity and specificity because tumor cells in general have nonspecific affinity to porphyrins (Dougherty, T. J., Gomer, C., Henderson, B. W., Jori, G., Kessel, D., Kprbelik, M., Moan, J., Peng, Q., Photodynamic Therapy, J. Natl. Cancer Inst. 90, 889-905, 1998; Schmidt-Erfurth, U., Diddens, H., Birngruber, R., Hasan, T., Photodynamic Targeting of Human Retinoblastoma Cells Using Covalent Low Density Lipoprotein Conjugates, Br. J. Cancer, 75, 54-61, 1997; Finber, V. H., Guo, H. H., Lu, Z. H., Peiper, S. C., Expression of Chemokine Receptors by Endothelial Cells: Detection by Intravital Microscopy Using Chemokine-Located Fluorescent Microspheres, Methods in Enzymology, 288, 148-158, 1997). Although the mechanism of porphyrin retention by tumors is not well understood, the balance between lipophilicity and hydrophilicity is recognized as an important factor (Henderson, B. W., Bellnier, D. A., Greco, W. R., Sharma, A., Pandey, R. K., Vaughan, L. A., Weishaupt, K. R., Dougherty, T. J., An in vivo Comparative Structure-Activity Relationship for a Congeneric Series of Pyropheophorbide Derivatives as Photosensitizers for Photodynamic Therapy, Cancer Research, 57, 4000-4007, 1997 and references therein; Pandey, R. K., Sumlin, A. B., Potter, W. R., Bellnier, D. A., Henderson, B. W., Constantine, S., Aoudia, M., Rodgers, J. A. J., Smith, K. M., Dougherty, T. J., Alkyl Ether Analogs of Chlorophyll A Derivatives: Synthesis, Photophysical Properties and Photodynamic Efficacy, Photochemistry and Photobiology, 64, 194-204, 1996; Zheng, G., Potter, W. R., Sumlin, A., Dougherty, T. J., Pandey, R. K., Photosensitizers Related to Purpurin-18-N-Alkylimides: A Comparative in vivo Tumoricidal Ability of Ester Versus Amide Functionalities, Bioorg. Med. Chem. Lett., 10, 123-127, 2000; Rungta, A., Zheng, G., Missert, J. R., Potter, W. R., Dougherty, T. J., Pandey, R. K., Purpurinimides as Photosensitizers: Effect of the Presence and Position of the Substituents in the in vivo Photodynamic Efficacy, Bioorg. Med. Chem. Lett., 10, 1463-1466, 2000).
Some attempts have been made to direct photosensitizers to known cellular targets by creating a photosensitizer conjugate, where the other molecule is a ligand that is specific for the target. For example, to improve localization to cell membranes cholesterol (Hombrecher, H. K., Schell, C., Thiem, J. Synthesis and Investigation of Galactopyranosyl-Cholesteryloxy Substituted Porphyrin, Bioorg. Med. Chem. Lett., 6:1199-1202, 1999) and antibody-conjugates have also been prepared to direct photosensitizers to specific tumor antigens (Donald, P. J., Cardiff, R. D., He, D., Kendell, K., Monoclonal Antibody-Porphyrin Conjugate for Head and Neck Cancer; the Possible Magic Bullet, Otolaryng Head Neck Surg., 105:781-787, 1991; Vrouenraets, M. B., Visser, G. W. M., Stewart, F. A., et al., Development of Meta-tetrahydroxyphenylchlorin-monoclonal Antibody Conjugate for Photoimmunotherapy, Cancer Res., 59:1505-1513, 1999). Certain chemotherapeutic agents have also been attached to porphyrin chromophores to increase the lethality of the PDT treatment (Karagianis, G., Reiss, J. A., Marchesini, R., et al., Biophysical and Biological Evaluation of Porphyrin-Bisacridine Conjugates, Anti-Cancer Drug Design 11:205-220, 1996). Certain protein- and microsphere-conjugates were made to improve the pharmacology of the compounds (Bachor, B. S., Shea, C. R., Gillies, R., Hasan, T., Photosensitized Destruction of Human Bladder Carcinoma Cells Treated with Cholrine6-Conjugates Microspheres, Proc. Natl. Acad. Sci., USA 88:1580-1584, 1991). These strategies seldom work well because the pharmacological properties of both compounds are drastically altered (MacDonald, I. J., Dougherty, T. J., Baqsic Principles of Photodynamic Therapy, J. Porphyrins Phthalocyanines, 5:105-129, 2001).
Since oligosaccharides play essential roles in molecular recognition, (Engvall, E., Enzyme Immunoassay ELISA and EMIT, Methods in Enzymology, 70, 419-439, 1980) porphyrins with sugar moieties should not only have good aqueous solubility but also possible specific membrane interaction. In recent years, various glycoconjugated porphyrins have been reported as potential photosensitizers; most of them are based on tetraphenylporphyrin (TPP) analogs (Chen, Y., Jain, R. K., Chandrasedkaran, E. V., Matta, K. L., Use of Sialylated or Sulfated Derivatives and Acrylamide Copolymers of Gal Beta 1,3GalNAc Alpha- and GalNAc Alpha- to Determine the Specificities of Anti-T and Anti-Tn Antibody Levels in Cancer Patients, Glycoconjugate J., 12, 55-62, 1995). However, none of them are known to show any specific cellular target, therefore, so far, no target-based rationally designed PDT agents based on this concept has been reported.
The galectins are a family of animal lectins defined by a highly conserved 15-kDa carbohydrate recognition domain (CRD) showing affinity for β-galactoside (Morgan, J., Potter, W. R., Oseroff, A. R., Comparison of Photodynamic Targets in a Carcinoma Cell Line and its Mitochondrial DNA-Deficient Derivatives, Photochem. Photobiol., 70, 747-757, 1999). Because galectins are involved in the modulation of cell adhesion, cell growth, immune response and angiogenesis, it is clear that changes in their expression might have a critical role in tumor progression. Galectin-1 (Gal-1) is a prototype, dimeric galectin with two identical CRDs, and its expression is known to correlate with the degree of malignancy in rat thyroid cell lines transformed with several cellular or viral oncogenes (MacDonald, I., Morgan, J., Bellnier, D. A., Paszkiewicz, G. M., Whitaker, J. E., Litchfield, D. J., Dougherty T. J., Subcellular Localization Patterns and their Relationship to Photodynamic Activity of Pyropheophorbide-a Derivatives, Photochem. Photobiol., 70, 789-997, 1999). Gal-1 mRNA levels increase 20 fold in low tumorigenic and up to 100-fold in high tumorigenic cells. These observations are consistent with those observed in human tumors (Kozyrev, A. N., Suresh, V., Das, S., Senge, M. O., Shibata, M., Dougherty, T. J., Pandey, R. K., Syntheses and Spectroscopic Studies of Novel Chlorins with Fused Quinoxaline or Benzimadazole Ring Systems and the Related Dimers with Extended Conjugation, Tetrahedron, 56, 3353-3364, 2000).
Furthermore, Gal-1 null mutant mice are found to be relatively healthy (Chiariotti, L., Salvatore, P., Benvenuto, G., Bruni, C. B., Control of Galectin Gene Expression, Biochimie, 81, 381-388, 1999; Salvatore, P., Benvenuto, G., Pero, R., Lembo, F., Bruni, C. B. and Chiaritti, L., Galectin-1 Gene Expression and Methylation State in Human T Leukemia Cell Lines, International Journal of Oncology, 17:1015-1018, 2000). Galectin-1 and galectin-3 are expressed in many epithelial tumors such as colon, thyroid, and breast carcinoma. However, there are still controversies whether galectin-3 promotes the metastatic potential and correlates with the poorly differentiated morphology or not. For example, the expression of galectin-3 inversely correlated with metastatic potential in breast and thyroid carcinoma, while in another study over-expression of galectin-3 conferred an increased metastatic potential to low metastatic cells in mouse melanoma and fibrosarcoma cells. It has recently been shown that the level of galectin expression increases and correlates with the neoplastic progression of colon carcinoma (Chiariotti, L., Salvatore, P., Benvenuto, G., Bruni, C. B., Control of Galectin Gene Expression, Biochimie, 81, 381-388, 1999; Salvatore, P., Benvenuto, G., Pero, R, Lembo, F., Bruni, C. B. and Chiaritti, L., Galectin-1 Gene Expression and Methylation State in Human T Leukemia Cell Lines, International Journal of Oncology, 17:1015-1018, 2000).
For the development of more effective cancer therapies it is important to have a better understanding of the molecular mechanisms that control invasion and metastases. In this regard, pathological interactions between cancer cells and the basement membrane (BM), a specialized extracellular matrix, have been extensively investigated (Castronovo, V., Laminin Receptors and Laminin-Bindign Proteins During Tumor Invasion and Metastasis, Invasion Metastasis, 13:1-30, 1993). The BM constitutes a barrier that cancer cells must cross several times during dissemination (Liotta, L. A., Tumor Invasion and Metastasis-Role of the Extracellular Matrix: Rhoads Memorial Award Lecture, Cancer Res., 46:1-7, 1986). Interactions between cancer cells and laminin, the main BM glycoprotein, are critical for successful invasion of these barriers. Several cell surface molecules have been described as laminin-binding proteins. Among these a few members of the galectin family also exhibit an altered pattern of expression in invasive and metastatic cancer cells (Castronovo, V., Laminin Receptors and Laminin-Bindign Proteins During Tumor Invasion and Metastasis, Invasion Metastasis, 13:1-30, 1993; Andre, S., Kojima, S., Yamazaki, N., Fink, S., Kaltner, H., Kaysev, K., Gabius, H. J., Galectin-1 and -3 and their Ligands in Tumor Biology, J. Cancer Res Clin Oncol., 125:461-474, 1999). Gal-1 and Gal-3 the two galactose-specific lectins bind laminin through its poly-N-acetyl-lactosamine residue. A high expression of Gal-1 and decreased expression of Gal-3 was found in uterine adenocarcinoma (Van Den Brule, F. A., Berchuck, A., Bast, R. C., Deprez, M., Liu, F. T., Cooper, D. N. W., Pieters, C., Bosel, M. E., and Castronovo, V., Expression of the 67-kD Laminin Receptor, Galectin-land Galectin-3 in Advanced Human Uterine Adenocarcinoma, Human Pathology, 27:1185-1191, 1996).
Analyses of the carbohydrate binding sites in Gal-1 and Gal-3 show that both galectins have a pronounced specificity for the Gal(β1-4)- and Gal(β1-3)GlcNAc sequences with no apparent affinity for GlcNAc residues. The binding specificity for Fal moiety is due to hydrogen bond interactions between its C4-hydroxy group and His 44, Asn 46 and Arg 48 residues that are conserved in all galectins. The van der Waals contact of Trp 68 with the Gal moiety as well as the hydrogen bonding of C6-hydroxy group to Gal-1 also contribute to binding (Rini, J. M., Lectin Structure, Annu. Rev. Biophys. Biomol. Struct. 24, 551-577, 1995).
Since our objective has included development of target specific photosensitizers as therapeutic agents for photodynamic therapy, we were interested in establishing a general synthetic route for the preparation of β-galactoside based long wavelength absorbing photosensitizers as Gal-1 recognizing agents. Our study was aimed at determining the effect of the presence of carbohydrate moieties on tumor selectivity and to explore the viability of this approach in converting an inactive compound with the required photophysical properties into an active therapeutic drug.